The most important barrier for efficient expression of foreign or native genes in any organism many times resides at the level of mRNA translation. As stability of mRNA is a major determinant of gene expression. According to the canonical model, the main signal for the ribosome to land on mRNA and start protein synthesis is the Shine-Dalgarno sequence, a purine-rich region located upstream of the start codon and complementary to the 3′-terminal sequence AUCACCUCCUUA (SEQ ID NO 1) (antiSD) of the 16S rRNA is thought that the SD-antiSD interaction directs the initiation codon to the P site of the 30S subunit at the first step of translation initiation, and that the efficiency of initiation and, eventually, of overall translation depends on the degree of complementarity of the two sequences: the tighter the SD duplex is, the more stable is the initiation complex and the higher the level of translation.
The 5′ untranslated region (5′ UTR) (also known as a Leader Sequence or Leader RNA) is the region of an mRNA that is directly upstream from the initiation codon. This region is important for the regulation of translation of a transcript by differing mechanisms in viruses, prokaryotes and eukaryotes. While called untranslated, the 5′ UTR or a portion of it is sometimes translated into a protein product. This product can then regulate the translation of the main coding sequence of the mRNA.
In many other organisms, however, the 5′ UTR is completely untranslated, instead forming complex secondary structure to regulate translation. The 5′ UTR has been found to interact with proteins relating to metabolism and proteins translate sequences within the 5′ UTR. In addition, this region has been involved in transcription regulation, such as the sex-lethal gene in Drosophila. The 5′ UTR begins at the transcription start site and ends one nucleotide (nt) before the initiation codon (usually AUG) of the coding region. In prokaryotes, the length of the 5′ UTR tends to be 3-10 nucleotides long while in eukaryotes it tends to be anywhere from 100 to several thousands nucleotides long. For example, the ste11 transcript in Schizosaccharomyces pombe has a 2273 nucleotide 5′ UTR while the lac operon in Escherichia coli only has 7 nucleotides in its 5′ UTR The differing sizes are likely due to the complexity of the eukaryotic regulation which the 5′ UTR holds, as well as the larger preinitiation complex which must form to begin translation.
The prokaryotic 5′ UTR contains a ribosome binding site (RBS), also known as the Shine Dalgarno sequence (AGGAGGU) which is usually 3-10 base pairs upstream from the initiation codon. As the 5′ UTR has a high GC content, secondary structures often occur within it. Hairpin loops are one such secondary structure that can be located within the 5′ UTR. These secondary structures also impact the regulation of translation. In prokaryotes, the initiation of translation occurs when IF-3 along with the 30S ribosomal subunit bind to the Shine-Dalgarno sequence of the 5′ UTR. This then recruits many other proteins that such as the 50S ribosomal subunit that allows for translation to begin.
Each of these steps regulates the initiation of translation. Translation machineries of Bacillus species are quite specific and require homologous ribosome binding sites RBS. The RBS usually contains a sequence GGAGG. It has an average free energy for binding is about −17 kcal/mol at 3′end of the 16S ribosomal RNA. Supplying an efficient Bacillus RBS sequence to the gene of interest is one solution, but it does not always work because the secondary structure around the translation initiation site also plays a pivotal role in determining translation efficiency. The sequence GGAGG in the RBS is usually highly conserved; and the spacer region between the GGAGG sequence and initiation codon is approximately eight bases long and rich in A and U nucleotides.
The absence of G and C residues around the RBS is thought to be optimal for ribosome binding. The crystals produced by Bacillus thuringiensis (Bt) mainly consist of Cry proteins, most of which are toxic for specific insects and consequently B. thuringiensis has been widely and successfully used as a biopesticides for more than 50 years. The crystal inclusion can account for up to 25% of the dry weight of B. thuringiensis cells. The mechanism for the massive expression of Cry proteins in B. thuringiensis has been investigated and involves numerous factors: transcriptional regulation, cry gene copy number, the stability of cry gene mRNA, and the accumulation and crystallization of Cry proteins.
Structure-based protein engineering of Cry toxins may direct the search for variants with broader susceptible species spectra, optimal potency, and stability properties. Cry2Aa is among an unusual subset of Cry proteins possessing broad insect species specificity by exhibiting high specific activity against two insect orders, Lepidoptera and Diptera. It is lethal to more lepidopteran species than the Cry1 toxins deployed against agriculturally important Lepidoptera and exhibits a low level of cross resistance in Cry1A-resistant insects. Also, the mode of action of Cry2Aa may be distinct from that of other Cry toxins. Thus, it could serve as a platform for the design of Cry toxins with broader susceptible species spectra and minimal Cry1A-derived cross resistance in the field.
Cry2A protein is of smaller mass having no C-terminal crystallization domain like that in the 130-140 kDa Cry proteins (e.g Cry1). The massive accumulation or crystallization of these Cry proteins generally requires the presence of additional proteins encoded by genes in the same operon. Additional protein of small size (29 kDa), have no insect toxicity and are not the main components of the crystals; rather, it enhances the accumulation or crystallization of their accompanying Cry protein. Consequently, it is described as an accessory proteins or helper protein. Helper protein is encoded by the orf1 and orf2 genes in the cry2A operon. Orf2 is necessary for the crystallization of Cry2A. It contains 11 tandem repeats of a 15/16 amino acid motif that is acidic in nature. Orf2 and Cry2A can be co-precipitated, evidence of interaction between the two proteins Indeed, Orf2 serves as a crystallization factor by interacting with the Cry2A protein, possibly acting as a template or scaffold.
To further investigate the role of Orf1 and Orf2 in Cry2A synthesis for potential applied use, we studied the effects of expressing cry2A alone or together with orf1 and orf2 by using the bioinformatics approach and experimental assessment. In this study, cyt1A promoter combined with the STAB-SD sequence (cyt1A-p/STAB-SD) was used as chimeric cyt1A-p/STAB-SD expression system has been shown to significantly improve synthesis of several Cry proteins and Bin toxins. Furthermore, the length and composition of the spacer region (RBS-ATG) was varied to scrutinize its effect on efficient production of Cry2Ac.